Oil-in-water emulsion

ABSTRACT

An oil-in-water emulsion comprising hydrophobin and oil wherein the oil phase has an iodine value of greater than 40 characterised in that the ratio of hydrophobin to oil is greater than 30 g/litre and less than 140 g/litre is provided. A product comprising such an oil-in-water emulsion is also provided.

TECHNICAL FIELD OF THE INVENTION

The present invention is directed to oil-in-water (o/w) emulsions. Inparticular, the present invention is directed to oil-in-water emulsionsthat are resistant to oxidisation.

BACKGROUND TO THE INVENTION

A wide variety of consumer goods contain oil-in-water emulsionsincluding cosmetic preparations (eg skin creams, moisturisers, lotions,and hair and skin conditioning agents) and food products (eg dressings,ice creams, mayonnaises, spreads and sauces). The physio-chemicalproperties of the emulsions are critical for ensuring consumeracceptance of these products and furthermore the stability of theemulsion and of the ingredients therein is vital for ensuring theshelf-life of such products.

There are a number of mechanisms that degrade the quality of a productcomprising an oil-in-water emulsion. Flocculation is the process bywhich particles in the emulsion are caused to clump together which maythen float to the top of the continuous phase or settle to the bottom ofthe continuous phase. Creaming is the migration of a substance in anemulsion, under the influence of buoyancy, to the top of a sample whilethe particles of the substance remain separated. Breaking andcoalescence is where the particles coalesce and form a layer within thecontinuous phase. Unstable emulsions are particularly susceptible tothese mechanisms and suffer a break down in the physio-chemicalstructure of the emulsion and the loss of the beneficial propertiesrequired by consumers. The quality of a product comprising anoil-in-water emulsion can be further affected through the degradation ofthe oil. Oxidation is one such process that may cause degradation andcan lead to rancidity and the loss of important functional ingredients.European Patent Application published as EP 1 978 824 discloses anaerated composition having an overrun of at least 10% and comprisingwater and an emulsified fat phase wherein the composition compriseshydrophobin. Examples 1 to 4 of EP 1 978 824 comprise Olive Oil which isnotorious for its sensitivity to oxidisation. There therefore remains aneed for oil-in-water emulsions with improved shelf-lives that areresistant to oxidisation of oil therein.

Tests and Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (eg colloid chemistry).

Oil

As used herein the term “oil” is used as a generic term for lipids,fats, or any mixture thereof, either pure or containing compounds insolution. Oils can also contain particles in suspension.

Lipids

As used herein the term “lipids” is used as a generic term for longchain fatty acids or long chain alcohols wherein the term “long chain”is used as a generic term for 12 carbon atoms or more.

Fats

As used herein the term “fats” is used as a generic term for compoundscontaining more than 80% triglycerides. They can also containdiglycerides, monoglycerides and free fatty acids. In common language,liquid fats are often referred to as oils but herein the term fats isalso used as a generic term for such liquid fats. Fats include: plantoils (for example: Apricot Kernel Oil, Arachis Oil, Arnica Oil, ArganOil, Avocado Oil, Babassu Oil, Baobab Oil, Black Seed Oil, BlackberrySeed Oil, Blackcurrant Seed Oil, Blueberry Seed Oil, Borage Oil,Calendula Oil, Camelina Oil, Camellia Seed Oil, Castor Oil, CherryKernel Oil, Cocoa Butter, Coconut Oil, Corn Oil, Cottonseed Oil, EveningPrimrose Oil, Grapefruit Oil, Grapeseed. Oil, Hazelnut Oil, HempseedOil, Jojoba Oil, Lemon Seed Oil, Lime Seed Oil, Linseed Oil, Kukui NutOil, Macadamia Oil, Maize Oil, Mango Butter, Meadowfoam Oil, Melon SeedOil, Moring a Oil, Olive Oil, Orange Seed Oil, Palm Oil, Papaya SeedOil, Passion Seed Oil, Peach Kernel Oil, Plum Oil, Pomegranate Seed Oil,Poppy Seed Oil, Pumpkins Seed Oil, Rapeseed (or Canola) Oil, RedRaspberry Seed Oil, Rice Bran Oil, Rosehip Oil, Safflower Oil,Seabuckthorn Oil, Sesame Oil, Soyabean Oil, Strawberry Seed Oil,Sunflower Oil, Sweet. Almond Oil, Walnut Oil, Wheat Germ Oil); fish oils(for example: Sardine Oil, Mackerel Oil, Herring Oil, Cod-liver Oil,Oyster Oil); animal oils (for example: Conjugated Linoleic Acid); orother oils (for example: Paraffinic Oils, Naphthenic Oils, AromaticOils, Silicone Oils); or any mixture thereof.

Iodine Value

As used herein the term “iodine value” is used as a generic term for themeasure of the unsaturation of oil and is expressed in terms of thenumber of centigrammes of iodine absorbed per gramme of sample (% iodineabsorbed). The higher the iodine number, the more unsaturated doublebonds are present in oil and hence the more prone the oil is tooxidisation via the double bond. Iodine value is determined using theWijs Method as provided in the American Oil Chemists' Society (AOCS)Official Method Tg 1a-64, pages 1-2, Official Methods and RecommendedPractices of the American Oil Chemists' Society, Second Edition, editedby D. Firestone, AOCS Press; Champaign, 1990, method Revised 1990).

Calculation of Ratio of Hydrophobin to Oil

As used herein the term “ratio of hydrophobin to oil” is defined as themass of hydrophobin (in grammes) relative to the volume of the oil (inlitres) in the oil-in-water emulsion. The ratio of hydrophobin to oil istherefore expressed as:

Total mass of Hydrophobin in emulsion (grammes):Total volume of oil inemulsion (litres)=g/litre

Calculation of Ratio of Oil to Water

As used herein the term “ratio of oil to water” is defined as the volumeof oil (in millilitres) relative to the volume of the water (inmillilitres) in the oil-in-water emulsion. The ratio of oil to water istherefore expressed as:

(Total volume of oil in emulsion (millilitres)/Total volume of water inemulsion (millilitres))×100=v/v %

Oil-in-Water Emulsion

As used herein the term “oil-in-water emulsion” is used as a genericterm for a mixture of two immiscible phases wherein an oil (dispersedphase) is dispersed in an aqueous solution (the continuous phase).

Food Products

As used herein the term “food products” is used as a generic term forproducts and ingredients taken by the mouth, the constituents of whichare active in and/or absorbed by the gastrointestinal tract with thepurpose of nourishment of the body and its tissues, refreshment andindulgence, which products are to be marketed and sold to customers forconsumption by humans. Examples of food products are tea, includingprecursors thereof; spreads; ice cream; frozen fruits and vegetables;snacks including diet foods and beverages; condiments; dressings; andculinary aids. Food products may particularly bring any of the followingbenefits: healthy metabolism; life span extension; optimal growth anddevelopment; optimal gastrointestinal tract function; avoidance ofmetabolic syndrome and insulin resistance; avoidance of dyslipidemias;weight control; healthy mineral metabolism; immune health; optimal eyehealth; avoidance of cognitive impairment and memory loss; hair and skinhealth; beauty; and excellent taste and smell.

Spreads

As used herein the term “spreads” is used as a generic term for oil andwater, containing emulsion, for instance a margarine type spread.Advantageously a spread has a pH of 4.8-6.0. The pH can be measured bymelting the spread, separating the molten fat phase from the water phaseand measuring the pH of the water phase.

Spreads of the invention may comprise other ingredients commonly usedfor spreads, such as flavouring ingredients, thickeners, gellationagents, colouring agents, vitamins, emulsifiers, pH regulators,stabilizers etc. Common amounts of such ingredients as well as suitableways to prepare margarines or spreads are well-known to the skilledperson.

Dressings

As used herein the term “dressings” is used as a generic term for oiland water containing emulsion, for instance vinaigrette andsalad-dressing type compositions.

Aeration

The term “aerated” means that gas has been intentionally incorporatedinto the product, such as by mechanical means. The gas can be any gas,but is preferably, particularly in the context of food products, afood-grade gas such as air, nitrogen or carbon dioxide. The extent ofaeration is typically defined in terms of “overrun”. In the context ofthe present invention, % overrun is defined in volume terms as:

Overrun=((volume of the final aerated product−volume of the mix)/volumeof the mix)×100

The amount of overrun present in the product will vary depending on thedesired product characteristics. For example, the level of overrun inconfectionery such as mousses can be as high as 200 to 250%. The levelof overrun in some chilled products, ambient products and hot productscan be lower, but generally over 10%, e.g. the level of overrun inmilkshakes is typically from 10 to 40%.

Hydrophobins

Hydrophobins are a well-defined class of proteins (Wessels, 1997, Adv.Microb. Physio. 38: 1-45; Wosten, 2001, Annu Rev. Microbiol. 55:625-646) capable of self-assembly at a hydrophobidhydrophilic interface,and having a conserved sequence:

(SEQ ID No. 1) Xn-C-X5-9-C-C-X11-39-C-X8-23-C-X5-9-C-C-X6-18-C-Xmwhere X represents any amino acid, and n and m independently representan integer. Typically, a hydrophobin has a length of up to 125 aminoacids. The cysteine residues (C) in the conserved sequence are part ofdisulphide bridges. In the context of the present invention, the termhydrophobin has a wider meaning to include functionally equivalentproteins still displaying the characteristic of self-assembly at ahydrophobic-hydrophilic interface resulting in a protein film, such asproteins comprising the sequence:

(SEQ ID No. 2) Xn-C-X1-50-C-X0-5-C-X1-100-C-X1-100-C-X1-50-C-X0-5-C-X1-50-C-Xmor parts thereof still displaying the characteristic of self-assembly ata hydrophobic-hydrophilic interface resulting in a protein film. Inaccordance with the definition of the present invention, self-assemblycan be detected by adsorbing the protein to Teflon and using CircularDichroism to establish the presence of a secondary structure (ingeneral, a-helix) (De Vocht et al., 1998, Biophys. J. 74: 2059-68).

The formation of a film can be established by incubating a Teflon sheetin the protein solution followed by at least three washes with water orbuffer (Wosten et al., 1994, Embo. J. 13: 5848-54). The protein film canbe visualised by any suitable method, such as labeling with afluorescent marker or by the use of fluorescent antibodies, as is wellestablished in the art. m and n typically have values ranging from 0 to2000, but more usually m and n in total are less than 100 or 200. Thedefinition of hydrophobin in the context of the present inventionincludes fusion proteins of a hydrophobin and another polypeptide aswell as conjugates of hydrophobin and other molecules such aspolysaccharides.

Hydrophobins identified to date are generally classed as either class Ior class II. Both types have been identified in fungi as secretedproteins that self-assemble at hydrophobilic interfaces into amphipathicfilms. Assemblages of class I hydrophobins are relatively insolublewhereas those of class II hydrophobins readily dissolve in a variety ofsolvents.

Hydrophobin-like proteins have also been identified in filamentousbacteria, such as Actinomycete and Steptomyces sp. (WO01/74864). Thesebacterial proteins, by contrast to fungal hydrophobins, form only up toone disulphide bridge since they have only two cysteine residues. Suchproteins are an example of functional equivalents to hydrophobins havingthe consensus sequences shown in SEQ ID Nos. 1 and 2, and are within thescope of the present invention.

The hydrophobins can be obtained by extraction from native sources, suchas filamentous fungi, by any suitable process. For example, hydrophobinscan be obtained by culturing filamentous fungi that secrete thehydrophobin into the growth medium or by extraction from fungal myceliawith 60% ethanol. It is particularly preferred to isolate hydrophobinsfrom host organisms that naturally secrete hydrophobins. Preferred hostsare hyphomycetes (e.g. Trichoderma), basidiomycetes and ascomycetes.Particularly preferred hosts are food grade organisms, such asCryphonectria parasitica which secretes a hydrophobin termed cryparin(MacCabe and Van Alfen, 1999, App. Environ. Microbiol 65: 5431-5435).

Alternatively, hydrophobins can be obtained by the use of recombinanttechnology. For example host cells, typically micro-organisms, may bemodified to express hydrophobins and the hydrophobins can then beisolated and used in accordance with the present invention. Techniquesfor introducing nucleic acid constructs encoding hydrophobins into hostcells are well known in the art. More than 34 genes coding forhydrophobins have been cloned, from over 16 fungal species (see forexample WO96/41882 which gives the sequence of hydrophobins identifiedin Agaricus bisporus; and Wosten, 2001, Annu Rev. Microbiol. 55:625-646). Recombinant technology can also be used to modify hydrophobinsequences or synthesise novel hydrophobins having desired/improvedproperties.

Typically, an appropriate host cell or organism is transformed by anucleic acid construct that encodes the desired hydrophobin. Thenucleotide sequence coding for the polypeptide can be inserted into asuitable expression vector encoding the necessary elements fortranscription and translation and in such a manner that they will beexpressed under appropriate conditions (e.g. in proper orientation andcorrect reading frame and with appropriate targeting and expressionsequences). The methods required to construct these expression vectorsare well known to those skilled in the art.

A number of expression systems may be used to express the polypeptidecoding sequence. These include, but are not limited to, bacteria, fungi(including yeast), insect cell systems, plant cell culture systems andplants all transformed with the appropriate expression vectors.Preferred hosts are those that are considered food grade—‘generallyregarded as safe’ (GRAS).

Suitable fungal species, include yeasts such as (but not limited to)those of the genera Saccharomyces, Kluyveromyces, Pichia, Hansenula,Candida, Schizosaccharomyces and the like, and filamentous species suchas (but not limited to) those of the genera Aspergillus, Trichoderma,Mucor, Neurospora, Fusarium and the like.

The sequences encoding the hydrophobins are preferably at least 80%identical at the amino acid level to a hydrophobin identified in nature,more preferably at least 95% or 100% identical. However, persons skilledin the art may make conservative substitutions or other amino acidchanges that do not reduce the biological activity of the hydrophobin.For the purpose of the invention these hydrophobins possessing this highlevel of identity to a hydrophobin that naturally occurs are alsoembraced within the term “hydrophobins”.

Hydrophobins can be purified from culture media or cellular extracts by,for example, the procedure described in WO01/57076 which involvesadsorbing the hydrophobin present in a hydrophobin-containing solutionto surface and then contacting the surface with a surfactant, such asTween 20, to elute the hydrophobin from the surface. See also Collen etal., 2002, Biochim. Biophys Acta. 1569: 139-50; Calonje et al., 2002,Can. J. Microbiol. 48: 1030-4; Askolin et al., 2001, Appl MicrobiolBiotechnol. 57: 124-30; and De Vries et al., 1999, Eur J. Biochem. 262:377-85.

Shelf-life

As used herein the term “shelf-life” is used as a generic term for thelength of time that a consumer product such as a food product may beconsidered suitable for sale or consumption. In particular, shelf-lifeis the time that products can be stored, during which the definedquality of a specified proportion of the goods remains acceptable underexpected (or specified) conditions of distribution, storage and display.In the instant case, shelf-life refers to the length of time that anoil-in-water emulsion maintains the physio-chemical properties criticalfor ensuring consumer acceptance of these products.

BRIEF DESCRIPTION OF THE INVENTION

We have now found that oil-in-water emulsions that are resistant tooxidisation may be obtained in formulations comprising certain amountsof hydrophobin and oil.

Accordingly, in a first aspect, the present invention provides anoil-in-water emulsion comprising hydrophobin and oil wherein the oil hasan iodine value of greater than 40 characterised in that the ratio ofhydrophobin to oil is greater than 30 g/litre and less than 140 g/litre,the oil-in-water emulsion having a ratio of oil to water of at least 1v/v %. Having conducted extensive research into the stabilisation ofoil-in-water emulsions and the prevention of degradation thereof we havefound that the advantage of the ratio of hydrophobin to oil is that insuch emulsions oxidisation of oil in the dispersed phase issignificantly reduced. Accordingly the ratio of hydrophobin to oil ispreferably greater than 35 g/litre, more preferably greater than 40g/litre, more preferably still greater than 60 g/litre and mostpreferably 80 g/litre. The total amount of hydrophobin used ispreferably less than 130 g/litre, more preferably less than 120 g/litre,more preferably still less than 100 g/litre.

As set out above, the hydrophobin may be a class I or a class IIhydrophobin, preferably a class II hydrophobin, more preferably thehydrophobin is HFBII.

The invention is capable of preventing the degradation of oxidisableoils and accordingly in a preferred embodiment the iodine value of theoil is greater than 60, more preferably greater than 90, more preferablystill greater than 120, most preferably greater than 140.

Particular oils are especially suitable for use according to theinvention and accordingly the oil is preferably selected from the groupconsisting of olive oil, corn oil, canola oil, soybean oil, sunfloweroil, linseed oil, and any mixture thereof.

Preferably the oil-in-water emulsion has an overrun of at least 20%,more preferably at least 40%, most preferably at least 80%. Theoil-in-water emulsion preferably has an overrun of at most 200%, morepreferably at most 150% and most preferably 100%.

Preferably the oil-in-water emulsion has a ratio of oil to water of atleast 1 v/v %, preferably at least 2 v/v % more preferably at least 5v/v %. The oil-in-water emulsion preferably has a ratio of oil to waterof at most 90 v/v %, more preferably at most 50 v/v %, more preferablystill at most 25 v/v % and most preferably at most 10 v/v %

In a second aspect, the present invention provides a food productcomprising an oil-in-water emulsion comprising an oil-in-water emulsioncomprising hydrophobin and oil wherein the oil phase has an iodine valueof greater than 40 characterised in that the ratio of hydrophobin to oilis greater than 30 g/litre and less than 140 g/litre, the oil-in-wateremulsion having a ratio of oil to water of at least 1 v/v %. Preferably,the oil-in-water emulsion has a ratio of oil to water of at least 2 v/v% more preferably at least 5 v/v %. The oil-in-water emulsion preferablyhas a ratio of oil to water of at most 90 v/v %, more preferably at most50 v/v %, more preferably still at most 25 v/v % and most preferably atmost 10 v/v %

The products are preferably food products, more preferably the foodproducts are selected from the group consisting of dressings, icecreams, mayonnaises, spreads and sauces.

DETAILED DESCRIPTION OF THE INVENTION Examples Hydrophobin

HFBII (Mw=7200 g.mol-1) solution from VTT Biotechnology Finland was usedfor all experiments. Unless otherwise stated, all water used for theexperiments was of Millipore quality.

Oils

Purified sunflower oil (SFO) was used for all experiments. Thecomposition and Iodine Value of the sunflower oil used is given in Table1

TABLE 1 Composition and Iodine Value of Sunflower Oil Fatty AcidComposition of Sunflower Oil (%) C6 0 C8 0 C10 0 C12 0 C14 0.1 C16 5.5C16:1 0.1 C18 4.7 C18:1 19.5 C18:2 68.5 C18:3 0.1 C20 0.3 C20:1 0.1 C220.9 C22:1 0 C24 0.2 Iodine Value = 135.8Preparation of Hydrophobin (HFB) and Whey Protein Isolate (WPI)solutions

pH adjusted Double Distilled Water (DDW) at pH 2, pH 3 and pH 7 wasprepared using 0.1M HCl or 0.1M NaOH. HFB or WPI was then incorporatedinto each pH adjusted DDW to a concentration of 0.2 wt % and the pH ofthe final solutions with each protein were adjusted with 0.1M HCl orNaOH to pH 2, 3 or 7. To these solutions, SFO was added and homogenizedas follows.

Preparation of Emulsions

Compositions according to Table 2 were first subjected to Ultraturrax at6, 500 RPM for 1 min, followed by 24,000 RPM for 10 min with continuousshaking of the beaker and then carefully transferred to Microfluidizer(MF) in order not to introduce unnecessary foam formation and subjectedto MF at 1000 bar for 5 min with ice to prevent temperature increase.Bottles of emulsions were then sealed and pasteurized at 80° C. for 10minutes and then allowed to cool.

TABLE 2 Composition of emulsions Comparative Example 1 Example A 0.2%HFB solution — 9.5 g 0.2% WPI solution 9.5 g — Sunflower Oil 0.5 g 0.5 g

Accelerated Oxidation Test

An accelerated oxidation test was performed on the pH 2, 3 and 7emulsions of Comparative Example 1 and Example A as provided below. Theaccelerated oxidation test was carried out over 42 days at 40° C.because the period and temperature are representative of a period offrom 9 to 12 months at a temperature of 20° C. The accelerated oxidationtest assesses the progress of oxidation through the measurement ofvolatile components resulting from oxidation and was performed asfollows:

-   1. 1 ml aliquots of Comparative Example 1 and Example A were    separated into individual vials.-   2. The air in the headspace of the vials was flushed out with    nitrogen gas.-   3. The vials were sealed with a cap with a rubber septum.-   4. All vials were placed in an incubator at 40° C. in the absence of    light for 42 days.-   5. After the 42 day experimental period the vials were removed from    the incubator and cooled in the absence of light to allow hexanal to    dissolve back into the emulsion. After cooling, the head space of    the vials was again flushed with nitrogen gas.-   6. Gas Chromatography (GC) was performed to detect volatile    components. During GC, the vials were heated to 60° C. to release    volatile components from the emulsion into the headspace which was    subsequently measured using GC. The levels of volatile components    detected using GS was expressed as peak area as calculated from the    chromatograms. Among the various volatiles, hexanal is the most    common representative of oxidation and the results therefore present    only Hexanal.

The results of the oxidation test for 42 days at 40° C. as shown inTables 3-5 show that HFB stabilized emulsions are less oxidized than WPIstabilized emulsions.

TABLE 3 Results of accelerated oxidation test of pH 2 emulsions after 42days Peak Area (μV/s) Comparative Example 1 2890470 Example A 81778

TABLE 4 Results of accelerated oxidation test of pH 3 emulsions after 42days Peak Area (μV/s) Comparative Example 1 2700784 Example A 153887

TABLE 5 Results of accelerated oxidation test of pH 7 emulsions after 42days Peak Area (μV/s) Comparative Example 1 2549786 Example A 1883759

1. An o/w emulsion comprising hydrophobin and oil wherein the oil phasehas an iodine value of greater than 40 characterised in that the ratioof hydrophobin to oil is greater than 30 g/litre and less than 140g/litre, the oil-in-water emulsion having a ratio of oil to water of atleast 1 v/v %.
 2. An o/w emulsion according to claim 1 wherein thehydrophobin is a class I or a class II hydrophobin, preferably a classII hydrophobin, more preferably HFBII.
 3. An o/w emulsion according toclaim 1 wherein the iodine value of the oil is greater than 60,preferably greater than 90, more preferably greater than 120, mostpreferably greater than
 140. 4. An o/w emulsion according to claim 1wherein the oil is selected from the group consisting of olive oil, cornoil, canola oil, soybean oil, sunflower oil, linseed oil, and anymixture thereof.
 5. An o/w emulsion according to claim 1 wherein theratio of hydrophobin to oil is greater than 40 g/litre, preferablygreater than 60 g/litre, more preferably greater than 60 g/litre andwherein the ratio of hydrophobin to oil is less than 130 g/litre,preferably less than 120 g/litre, more preferably less than 100 g/litre.6. A food product, comprising an oil-in-water emulsion comprisinghydrophobin and oil wherein the oil phase has an iodine value of greaterthan 40 characterised in that the ratio of hydrophobin to oil is greaterthan 30 g/litre and less than 140 g/litre, the oil-in-water emulsionhaving a ratio of oil to water of at least 1 v/v %.